Despite many recent advances in understanding the molecular and cellular biology of Mycobacterium tuberculosis (MTB), the rate-limiting step in nearly all experiments remains batch-culture growth and phenotyping - an imprecise process of modest throughput that masks the heterogeneity of responses and is largely unchanged in the last 100 years. Like all bacteria, Mycobacterium tuberculosis (MTB) is subjected to dynamic environmental stresses throughout its natural history. Faced with varied and changing environments, bacteria respond in diverse ways - and the breadth of these responses cannot be captured in batch culture. Here we propose to develop a novel and robust method, Parallel Microscopic Determination of Fitness (PMDF), to monitor bacterial growth and viability while preserving and recording phenotypic heterogeneity within the population. We have performed pilot experiments using time lapse phase and fluorescent microscopy to monitor growth of the soil saprophyte Mycobacterium smegmatis, following the independent fates of thousands of separate cell deposition events in each experiment. Our goal now is to adapt this experimental platform to MTB, with a series of proof-of-concept experiments. We will characterize the replication of geographically diverse clinical isolates of MTB as they respond over time to defined, disease- relevant environmental perturbations, and develop this approach to improve analysis of susceptibility to small molecule antitubercular agents.